Owing to recent development of genetic engineering and protein engineering, methods for obtaining proteins have been changing, and among others a method, in which a gene encoding a target protein is isolated and expressed in an appropriate host cell, has enabled acquisition of a large amount of the target protein. For such synthesis of a protein from a gene, overexpression of the gene in E. coli, an insect cell or an animal cell, or a cell-free protein synthesis method is applied.
In case of protein synthesis using an insect cell or a mammal cell, the obtained protein often has a regulated higher order structure to form an ordered 3D-structure, and is soluble. By such methods, however, the yield of the target protein is very low, complex purification procedures are required to recover such protein, and long time is necessary to obtain the target protein. Further, the obtained target protein is very limited in a quantity, and is expensive.
On the other hand, protein synthesis using E. coli is easy in handling, requires only short time to obtain the target protein, and is inexpensive. Consequently, a method using an E. Coli transfected with a gene coding for synthesis of the target protein is the mainstream of the protein synthesis nowadays and a production process is being established. However, many observations have been reported concerning proteins of higher organisms including a human, which are produced by an E. coli expression system, that an aggregate of insolubilized protein called as an inclusion body is generated intracellularly. Such insolubilized protein of an inclusion body, of course, is lacking its inherent function or ability, and does not show activity. Consequently, in an artificial protein production process, it is required to unravel an inclusion body, to reform the higher structure, and to convert the protein to an active form with an ordered 3D-structure. Namely a refolding procedure of an inclusion body is necessary.
Such refolding is an important technology, which can be applicable not only to a protein produced by E. coli but also to reactivation of a protein deactivated by thermal history or other causes, and has been studied broadly. Various methods have been proposed, but they have attained only low levels of refolding rates, or better results only accidentally for some limited proteins.
For example, a method, by which an insolubilized protein is solubilized using a protein modifier such as urea or guanidine hydrochloride, and then refolded gradually by removing the protein modifier, is widely used. The method is, however, not satisfactory, since it requires time-consuming selection of the conditions for spontaneous refolding of a protein, and there remain many proteins which can be refolded by the method. The method cannot meet the current requirements for large quantity productions of proteins.
Under such circumstances, a refolding method using a molecular chaperone has received increased attention. A molecular chaperone, formerly known as a heat-shock protein, is a group of proteins which are known as proteins with functions concerning refolding, membrane permeability, association, decomposition of a protein, their gene sequences are highly conserved from E. coli to a human. Many of molecular chaperones are generated when an organism is subjected to a heat shock, metabolic inhibition, heavy metal, viral infection, ischemia, etc., and function to protect the organism against such stress and shock to maintain its homeostasis. However, their refolding mechanism has not been yet clarified. As an artificial molecular, chaperone β-cyclodextrin or cycloamylase is used. It is described in J. Am. Chem. Soc., Vol. 117, (1995) 2373-2374, that when a denatured protein is mixed with a solution of such artificial chaperone, inclusion and removal by the artificial chaperone take place and during such process the protein is refolded. The method has been successful only for such limited proteins as carbonicanhydrase B, and it is expensive since it is not recyclable.
Japanese Patent Application Laid-Open No. 2005-029531 has disclosed a method using zeolite beta. By the method, an inclusion body is contacted with zeolite beta and adsorbed on it, and then the inclusion body is desorbed by a surfactant to refold the protein which molecular weight can be higher than 100,000. Successful refolding has been confirmed with not less than several proteins. However, a selection of a surfactant for a desorption process from an adsorbent, zeolite beta, has a crucial impact on a refolding rate, and complex condition setting is required to address a specific nature of a protein. The process becomes more complex than other conventional refolding processes.
[Patent Document 1] Japanese Patent Application Laid-Open No. 2005-029531
[Non-patent Document 1] J. Am. Chem. Soc., Vol. 117, (1995) 2373-2374